Obesity-induced inflammation plays an important role in the link between obesity and diseases such as cardiovascular disease and type 2 diabetes. The mechanism behind this relates to the wide range leukocytes found in adipose tissue that secrete numerous inflammatory mediators. Since the initial identification of macrophages in fat, the number of leukocyte types present in fat have greatly increased and now include most leukocyte types. Unfortunately, an integrated understanding of how these cells interact with each other and with adipocytes is lacking and limits progress in the field. We feel that this gap in our understanding is due in large part to the limited techniques available to assay inflammatory cells in fat. Furthermore, we feel that there are many dynamic cellular events that are occurring in fat but are not being captured. Therefore, the goal of this study is to design and implement a novel methodology for intravital microscopy on adipose tissue in mouse models of obesity. Being able to observe leukocyte interactions in fat can reveal the mechanisms involved in the initiation and maintenance of inflammation in obesity. This advance will be significant as it has the potential to unlock the mystery of how inflammation is generated in obesity. We will accomplish our goal by completing two aims. (1) To establish the methodology for intravital imaging of leukocytes in adipose tissue. This will employ two-photon confocal microscopy to generate 4D views of fat in live animals. (2) To evaluate the effects of obesity on T cell migration in relation to fat associated lymphoid clusters (FALCs). We will use intravital microscopy to test a model of obesity-induced induction of T cell flux out of FALCs and into fat to generate inflammation. At the end of the project, we hope to generate a new standardized tool for obesity researchers that can generate new models and provide a window into the dynamic properties of adipose tissue stromal cells. PUBLIC HEALTH RELEVANCE: With obesity, adipose tissue dysfunction contributes the development of diseases such as diabetes and metabolic syndrome. This is closely linked to the recruitment and activation of inflammatory cells to fat. Research in this area is currently limited due to few methods that are available to researchers and clinicians to study inflammatory cells in fat. To address this, we will develop a novel resource for obesity researchers to enable visualization of inflammatory cells in live adipose tissue.